OBJECTIVES: The objective of the research proposed below is to determine the mechanisms whereby the intracellular level of cyclic AMP is regulated. The studies will focus on the mechanisms utilized by cells in culture to regulate their responsiveness to catecholamines and prostaglandins. We have established at least four different experimental circumstances that alter cellular responsiveness to the hormones: (1) prior exposure to hormones reduces subsequent responsiveness, (2) cells in mitosis and early G1 have greatly reduced responsiveness to catecholamines, (3) cholera toxin induces a state of hyperresponsiveness to both catecholamines and prostaglandins, (4) glucocorticoids induce a selective increase in the responsiveness of cells to prostaglandins. Our specific objectives are to determine the molecular basis of these changes in the responsiveness of adenylate cyclase to hormones. We will attempt to identify alterations in the receptor, GTP binding protein or catalytic moieties of adenylate cyclase that account for the changes in responsiveness to the hormones. SPECIFIC AIMS: 1. To isolate and characterize the putative intracellular intermediate that promotes heterologous desensitization. 2. To determine if agonist-specific induction of the phosphorylation of unique membrane components occurs concomitant with desensitization and is lost concomitant with the return of hormonal responsiveness. 3. To determine if cells in mitosis and early G1 that exhibit minimal responsiveness to catecholamines bind less 125I-hydroxybenzylpindolol -suggesting a reduction in the number of beta-receptors. 4. To determine the effect of the "active" subunit of cholera toxin on the responsiveness of adenylate cyclase in membranes to isoproterenol and prostaglandin E1.